Three-component protein complexes spanning two membranes are universally spread among Gram-negative bacteria and have been implicated in such diverse range of transport functions as delivery of virulence factors into the hosts, secretion of signaling molecules and protection of bacterial cells against structurally diverse antimicrobial agents. A remarkable feature of these transporters is that the substrate transfer occurs across two membranes directly into external medium bypassing the periplasmic space. Special periplasmic proteins, which belong to the Membrane Fusion Protein (MFP) family, are responsible for the coupling of two membranes. The major objective of this project will be to investigate the mechanism of coupling of two membranes using the multidrug efflux transporter AcrAB-TolC from E. coli as a model complex. The AcrAB-TolC extrudes out of the cell a broad range of antimicrobial compounds including antibiotics, detergents, dyes and organic solvents. Located in the inner membrane AcrB transporter captures its substrates within phospholipid bilayer of inner membrane and transports them into external medium via the outer membrane channel, ToIC. The cooperation between AcrB and TolC is mediated by the MFP protein, AcrA. Biochemical studies suggested that AcrA might coordinate the function of the complex by bringing the inner and outer membranes into proximity. The applicants intend to test this hypothesis critically and to investigate the mechanism of AcrA. The specific aims are: (1) investigate if AcrA has differential affinity for the inner and outer membranes; (2) using limited proteolysis and chemical cross-linking, characterize the conformation of AcrA in vivo and in vitro; (3) characterize conformational transitions of AcrA in vitro; and (4) investigate physical and functional interactions between AcrA, AcrB and TolC in vitro. These studies are expected to yield a comprehensive picture of AcrA-mediated coupling of two membranes. These data will contribute to an understanding of the mechanism of multidrug efflux in Gram-negative bacteria and will illuminate the role of MFP proteins in the coordination of transport across two membranes.